Transplantation of gene-modified haematopoietic stem cells: Application and clinical considerations.

Autologous and allogeneic haematopoietic stem cell (HSC) transplantation has been performed in patients with various malignant and non-malignant haematological disorders for more than 50 years. Ex vivo gene modification of HSCs for autologous transplantation opens up new therapeutic avenues for genetic and infectious diseases. Major advances have been made over the last three decades with respect to gene modification of HSCs and transplantation strategies, ultimately culminating in the approval of two such therapies in Europe (Strimvelis for a rare primary immune deficiency, and LentiGlobin for beta-thalassaemia). Newer gene-modifying technologies and treatment regimens have also recently come to the fore, which hold great promise for the development of safer and more effective treatments. We provide an overview of the current state of gene-modified HSC therapies, highlighting success stories, limitations and important considerations for achieving successful translation of these therapies to the clinic.

Decreased NOX2 expression in the brain of patients with bipolar disorder: association with valproic acid prescription and substance abuse.

Neuroinflammation and increased oxidative stress are believed to contribute to the development of psychiatric diseases. Animal studies have implicated NADPH oxidases (NOX) as relevant sources of reactive oxygen species in the brain. We have analyzed the expression of NOX isoforms in post-mortem brain samples from patients with psychiatric disorders (schizophrenia, bipolar disorder) and non-psychiatric subjects. Two collections from the Stanley Medical Research Institute were studied: the Array Collection (RNA, 35 individuals per group), and a neuropathology consortium collection (paraffin-embedded sections, 15 individuals per group). Quantitative PCR analysis revealed expression of NOX2 and NOX4 in prefrontal cortex. No impact of psychiatric disease on NOX4 levels was detected. Remarkably, the expression of NOX2 was specifically decreased in prefrontal and cingulate cortices of bipolar patients, as compared with controls and schizophrenic patients. NOX2 expression was not statistically associated with demographic parameters and post-mortem interval, but correlated with brain pH. Immunostaining demonstrated that NOX2 was predominantly expressed in microglia, which was corroborated by a decrease in the microglial markers CD68 and CD11b in the cingulate cortex of bipolar disorder patients. The analysis of potentially confounding parameters showed association of valproic acid prescription and heavy substance abuse with lower levels of NOX2. Taken together, we did not observe changes of NOX2 in schizophrenic patients, but a marked decrease of microglial markers and NOX2 in the brain of bipolar patients. This might be an underlying feature of bipolar disorder and/or a consequence of valproic acid treatment and substance abuse.

[Cell transplantation: current treatments and future prospects]. / Transplantation cellulaire: traitements actuels et perspectives d'avenir.

Regenerative medicine aims to replace a body function or specific cell loss. It includes therapies at the forefront of modem medicine, issuing from translational biomedical research. Transplantation of organs and cells has revolutionized the management of patients for whom medical treatment is a failure. Unfortunately, organ shortage is limiting treatment possibility. As an example, among the 15,000 patients with type I diabetes in Switzerland, only approximately 30 can receive a pancreas or an islet transplant per year. Second example, 500 patients die each year in Switzerland from alcoholic cirrhosis because no treatment is available. Transplantation of islet cells, hepatocytes, mesenchymal stem cells or dopaminergic neurons represents hope fora therapy available for large populations of patients.

Reactive oxygen species: from health to disease.

Upon reaction with electrons, oxygen is transformed into reactive oxygen species (ROS). It has long been known that ROS can destroy bacteria and destroy human cells, but research in recent decades has highlighted new roles for ROS in health and disease. Indeed, while prolonged exposure to high ROS concentrations may lead to non-specific damage to proteins, lipids, and nucleic acids, low to intermediate ROS concentrations exert their effects rather through regulation of cell signalling cascades. Biological specificity is achieved through the amount, duration, and localisation of ROS production. ROS have crucial roles in normal physiological processes, such as through redox regulation of protein phosphorylation, ion channels, and transcription factors. ROS are also required for biosynthetic processes, including thyroid hormone production and crosslinking of extracellular matrix. There are multiple sources of ROS, including NADPH oxidase enzymes; similarly, there are a large number of ROS-degrading systems. ROS-related disease can be either due to a lack of ROS (e.g., chronic granulomatous disease, certain autoimmune disorders) or a surplus of ROS (e.g., cardiovascular and neurodegenerative diseases). For diseases caused by a surplus of ROS, antioxidant supplementation has proven largely ineffective in clinical studies, most probably because their action is too late, too little, and too non-specific. Specific inhibition of ROS-producing enzymes is an approach more promising of clinical efficacy.

NADPH oxidase elevations in pyramidal neurons drive psychosocial stress-induced neuropathology.

Oxidative stress is thought to be involved in the development of behavioral and histopathological alterations in animal models of psychosis. Here we investigate the causal contribution of reactive oxygen species generation by the phagocyte NADPH oxidase NOX2 to neuropathological alterations in a rat model of chronic psychosocial stress. In rats exposed to social isolation, the earliest neuropathological alterations were signs of oxidative stress and appearance of NOX2. Alterations in behavior, increase in glutamate levels and loss of parvalbumin were detectable after 4 weeks of social isolation. The expression of the NOX2 subunit p47(phox) was markedly increased in pyramidal neurons of isolated rats, but below detection threshold in GABAergic neurons, astrocytes and microglia. Rats with a loss of function mutation in the NOX2 subunit p47(phox) were protected from behavioral and neuropathological alterations induced by social isolation. To test reversibility, we applied the antioxidant/NOX inhibitor apocynin after initiation of social isolation for a time period of 3 weeks. Apocynin reversed behavioral alterations fully when applied after 4 weeks of social isolation, but only partially after 7 weeks. Our results demonstrate that social isolation induces rapid elevations of the NOX2 complex in the brain. Expression of the enzyme complex was strongest in pyramidal neurons and a loss of function mutation prevented neuropathology induced by social isolation. Finally, at least at early stages, pharmacological targeting of NOX2 activity might reverse behavioral alterations.

Generation and applications of human pluripotent stem cells induced into neural lineages and neural tissues.

Human pluripotent stem cells (hPSCs) represent a new and exciting field in modern medicine, now the focus of many researchers and media outlets. The hype is well-earned because of the potential of stem cells to contribute to disease modeling, drug screening, and even therapeutic approaches. In this review, we focus first on neural differentiation of these cells. In a second part we compare the various cell types available and their advantages for in vitro modeling. Then we provide a "state-of-the-art" report about two major biomedical applications: (1) the drug and toxicity screening and (2) the neural tissue replacement. Finally, we made an overview about current biomedical research using differentiated hPSCs.

The miR 302-367 cluster drastically affects self-renewal and infiltration properties of glioma-initiating cells through CXCR4 repression and consequent disruption of the SHH-GLI-NANOG network.

Glioblastoma multiforme (GBM) is the most common form of primary brain tumor in adults, often characterized by poor survival. Glioma-initiating cells (GiCs) are defined by their extensive self-renewal, differentiation, and tumor initiation properties. GiCs are known to be involved in tumor growth and recurrence, and in resistance to conventional treatments. One strategy to efficiently target GiCs in GBM consists in suppressing their stemness and consequently their tumorigenic properties. In this study, we show that the miR-302-367 cluster is strongly induced during serum-mediated stemness suppression. Stable miR-302-367 cluster expression is sufficient to suppress the stemness signature, self-renewal, and cell infiltration within a host brain tissue, through inhibition of the CXCR4 pathway. Furthermore, inhibition of CXCR4 leads to the disruption of the sonic hedgehog (SHH)-GLI-NANOG network, which is involved in self-renewal and expression of the embryonic stem cell-like signature. In conclusion, we demonstrated that the miR-302-367 cluster is able to efficiently trigger a cascade of inhibitory events leading to the disruption of GiCs stem-like and tumorigenic properties.

Primate-specific RFPL1 gene controls cell-cycle progression through cyclin B1/Cdc2 degradation.

Ret finger protein-like 1 (RFPL1) is a primate-specific target gene of Pax6, a key transcription factor for pancreas, eye and neocortex development. However, its cellular activity remains elusive. In this article, we report that Pax6-elicited expression of the human (h)RFPL1 gene in HeLa cells can be enhanced by in vivo p53 binding to its promoter and therefore investigated the hypothesis that hRFPL1 regulates cell-cycle progression. Upon expression in these cells, hRFPL1 decreased cell number through a kinase-dependent mechanism as PKC activates and Cdc2 inhibits hRFPL1 activity. hRFPL1 antiproliferative activity led to an increased cell population in G(2)/M phase and specific cyclin B1 and Cdc2 downregulations, which were precluded by a proteasome inhibitor. Specifically, cytoplasm-localized hRFPL1 prevented cyclin B1 and Cdc2 accumulation during interphase. Consequently, cells showed a delayed entry into mitosis and cell-cycle lengthening resulting from a threefold increase in G(2) phase duration. Given previous reports that RFPL1 is expressed during cell differentiation, its impact on cell-cycle lengthening therefore provides novel insights into primate-specific development.

Neural commitment of embryonic stem cells: molecules, pathways and potential for cell therapy.

The study of neuronal differentiation of embryonic stem cells has raised major interest over recent years. It allows a better understanding of fundamental aspects of neurogenesis and, at the same time, the generation of neurons as tools for various applications ranging from drug testing to cell therapy and regenerative medicine. Since the first report of human embryonic stem (ES) cells derivation, many studies have shown the possibility of directing their differentiation towards neurons. However, there are still many challenges ahead, including gaining a better understanding of the mechanisms involved and developing techniques to allow the generation of homogeneous neuronal and glial subtypes. We review the current state of knowledge of embryonic neurogenesis which has been acquired from animal models and discuss its translation into in vitro strategies of neuronal differentiation of ES cells. We also highlight several aspects of current protocols which need to be optimized to generate high-quality embryonic stem cell-derived neuronal precursors suitable for clinical applications. Finally, we discuss the potential of embryonic stem cell-derived neurons for cell replacement therapy in several central nervous system diseases.

Branched fungal beta-glucan causes hyperinflammation and necrosis in phagocyte NADPH oxidase-deficient mice.

Chronic granulomatous disease (CGD), a genetic disorder characterized by the absence of a functional phagocyte NADPH oxidase, is a severe immune deficiency. However, non-infectious hyperinflammation is a second hallmark of the disease. In CGD mouse models, sterile hyperinflammation can be induced by A. fumigatus cell wall preparations. In this study, we used subcutaneous injection of microbial cell walls and cell wall components to identify causes of CGD hyperinflammation and to characterize its histological features. Sterile cell wall preparations from fungi (A. fumigatus, C. albicans, S. cerevisiae), but not from bacteria (S. aureus, P. aeruginosa, E. coli), caused prolonged and severe skin inflammation in CGD mice. To identify fungal cell wall elements responsible for this process, we investigated microbial cell wall-derived monosubstances. Injection of beta(1-3)(1-6)-glucan induced severe hyperinflammation in CGD mice, while other fungal cell components [mannan, (1-3) beta-glucan] or bacterial cell wall components (lipopolysaccharide, lipoteichoic acid) caused no or only moderate inflammation. beta-glucan-induced hyperinflammation was predominantly due to a defect in termination of inflammation, as in the initial stage (2 days), the severity of inflammation and the extent of cell death were comparable in wild-type and CGD mice. At later stages (7 days), beta(1-3)(1-6)-glucan-induced inflammation had subsided in wild-type mice. In contrast, CGD mice showed persistent severe inflammation with central necrosis, containing abundant apoptotic and necrotic cells. In summary, branched fungal beta-glucan induces a severe inflammatory reaction in the absence of phagocyte NADPH oxidase. As opposed to the commonly perceived notion that reactive oxygen species are the cause of cell death, our results demonstrate that tissue necrosis can be caused by the absence of a superoxide-producing enzyme.

Scapuloperoneal syndrome type Kaeser and a wide phenotypic spectrum of adult-onset, dominant myopathies are associated with the desmin mutation R350P.

In 1965, an adult-onset, autosomal dominant disorder with a peculiar scapuloperoneal distribution of weakness and atrophy was described in a large, multi-generation kindred and named 'scapuloperoneal syndrome type Kaeser' (OMIM #181400). By genetic analysis of the original kindred, we discovered a heterozygous missense mutation of the desmin gene (R350P) cosegregating with the disorder. Moreover, we detected DES R350P in four unrelated German families allowing for genotype-phenotype correlations in a total of 15 patients carrying the same mutation. Large clinical variability was recognized, even within the same family, ranging from scapuloperoneal (n = 2, 12%), limb girdle (n = 10, 60%) and distal phenotypes (n = 3, 18%) with variable cardiac (n = 7, 41%) or respiratory involvement (n = 7, 41%). Facial weakness, dysphagia and gynaecomastia were frequent additional symptoms. Overall and within each family, affected men seemingly bear a higher risk of sudden, cardiac death as compared to affected women. Moreover, histological and immunohistochemical examination of muscle biopsy specimens revealed a wide spectrum of findings ranging from near normal or unspecific pathology to typical, myofibrillar changes with accumulation of desmin. This study reveals that the clinical and pathological variability generally observed in desminopathies may not be attributed to the nature of the DES mutation alone, but may be influenced by additional genetic and epigenetic factors such as gender. In addition, mutations of the desmin gene should be considered early in the diagnostic work-up of any adult-onset, dominant myopathy, even if specific myofibrillar pathology is absent.

Embryonic and adult stem cell-derived cardiomyocytes: lessons from in vitro models.

For years, research has focused on how to treat heart failure by sustaining the overloaded remaining cardiomyocytes. Recently, the concept of cell replacement therapy as a treatment of heart diseases has opened a new area of investigation. In vitro-generated cardiomyocytes could be injected into the heart to rescue the function of a damaged myocardium. Embryonic and/or adult stem cells could provide cardiac cells for this purpose. Knowledge of fundamental cardiac differentiation mechanisms unraveled by studies on animal models has been improved using in vitro models of cardiogenesis such as mouse embryonal carcinoma cells, mouse embryonic stem cells and, recently, human embryonic stem cells. On the other hand, studies suggesting the existence of cardiac stem cells and the potential of adult stem cells from bone marrow or skeletal muscle to differentiate toward unexpected phenotypes raise hope and questions about their potential use for cardiac cell therapy. In this review, we compare the specificities of embryonic vs adult stem cell populations regarding their cardiac differentiation potential, and we give an overview of what in vitro models have taught us about cardiogenesis.

Expression of NOX1, a superoxide-generating NADPH oxidase, in colon cancer and inflammatory bowel disease.

Reactive oxygen species (ROS) are at the centre of many physiological and pathological processes. NOX1, a ROS-producing NADPH oxidase, is highly expressed in the colon but its function in colonic physiology or pathology is still poorly understood. It has been suggested to play a role in host defence, but also in cell growth and possibly malignant transformation. In this study we characterized NOX1 expression in human colon samples derived from healthy control subjects and patients with colon cancer or inflammatory bowel disease (IBD). NOX1 mRNA expression was assessed by dot-blot hybridization, real-time PCR and in situ hybridization, using samples derived from surgical specimens from patients undergoing colon resection. In normal tissues, NOX1 expression was low in the ileum, intermediate in the right colon, and high in the left colon (p = 0.0056 right vs. left colon). NOX1 mRNA levels were not influenced by factors linked to colon tumourigenesis, such as age or sex. Moreover, there was no statistical difference in NOX1 expression between samples derived from adenomas, well differentiated or poorly differentiated colon adenocarcinomas. At a cellular level, NOX1 was highly expressed in colon epithelial cells, both within the crypts and on the luminal surface. In addition, a population of lymphocytes, particularly in the appendix, showed NOX1 expression. Lymphocytes in lesions of Crohn's disease and ulcerative colitis were also strongly positive for NOX1. In conclusion, NOX1 is an enzyme that is constitutively expressed in colon epithelium and is not associated with tumourigenesis. Its distribution in crypts and on the luminal surface, as well as its left-to-right gradient in the colon, suggests a role in host defence function. In addition to the known epithelial localization, we define lymphocytes as a novel site of NOX1 expression, where it may potentially be involved in the pathogenesis of inflammatory bowel diseases.

Functional specialization of calreticulin domains.

Calreticulin is a Ca2+-binding chaperone in the endoplasmic reticulum (ER), and calreticulin gene knockout is embryonic lethal. Here, we used calreticulin-deficient mouse embryonic fibroblasts to examine the function of calreticulin as a regulator of Ca2+ homeostasis. In cells without calreticulin, the ER has a lower capacity for Ca2+ storage, although the free ER luminal Ca2+ concentration is unchanged. Calreticulin-deficient cells show inhibited Ca2+ release in response to bradykinin, yet they release Ca2+ upon direct activation with the inositol 1,4,5-trisphosphate (InsP3). These cells fail to produce a measurable level of InsP3 upon stimulation with bradykinin, likely because the binding of bradykinin to its cell surface receptor is impaired. Bradykinin binding and bradykinin-induced Ca2+ release are both restored by expression of full-length calreticulin and the N + P domain of the protein. Expression of the P + C domain of calreticulin does not affect bradykinin-induced Ca2+ release but restores the ER Ca2+ storage capacity. Our results indicate that calreticulin may play a role in folding of the bradykinin receptor, which affects its ability to initiate InsP3-dependent Ca2+ release in calreticulin-deficient cells. We concluded that the C domain of calreticulin plays a role in Ca2+ storage and that the N domain may participate in its chaperone functions.

Heme histidine ligands within gp91(phox) modulate proton conduction by the phagocyte NADPH oxidase.

The membrane subunit of the phagocyte NADPH oxidase, gp91(phox), possesses a H(+) channel motif formed by membrane-spanning histidines postulated to coordinate the two heme groups forming the redox center of the flavocytochrome. To study the role of heme-binding histidines on proton conduction, we stably expressed the gp91(phox) cytochrome in human embryonic kidney 293 cells and measured proton currents with the patch clamp technique. Similar to its shorter homologue, NADPH oxidase homologue 1, which is predicted not to bind heme, gp91(phox) generated voltage-activated, pH-dependent, H(+)-selective currents that were reversibly blocked by Zn(2+). The gp91(phox) currents, however, activated faster, deactivated more slowly, and were markedly affected by the inhibition of heme synthesis. Upon heme removal, the currents had larger amplitude, activated faster and at lower voltages, and became sensitive to the histidine reagent diethylpyrocarbonate. Mutation of the His-115 residue to leucine abolished both the gp91(phox) characteristic 558-nm absorbance peak and voltage-activated currents, indicating that His-115 is involved in both heme ligation and proton conduction. These results indicate that the gp91(phox) proton channel is activated upon release of heme from its His-115 ligand. During activation of the oxidase complex, changes in heme coordination within the cytochrome might increase the mobility of histidine ligands, thereby coupling electron and proton transport.

Identification of BARD1 as mediator between proapoptotic stress and p53-dependent apoptosis.

The BRCA1-associated protein BARD1 is a putative tumor suppressor. We suggest that BARD1 is a mediator of apoptosis since (1) cell death in vivo (ischemic stroke) and in vitro is accompanied by increased levels of BARD1 protein and mRNA; (2) overexpression of BARD1 induces cell death with all features of apoptosis; and (3) BARD1-repressed cells are defective for the apoptotic response to genotoxic stress. The proapoptotic activity of BARD1 involves binding to and elevations of p53. BRCA1 is not required for but partially counteracts apoptosis induction by BARD1. A tumor-associated mutation Q564H of BARD1 is defective in apoptosis induction, thus suggesting a role of BARD1 in tumor suppression by mediating the signaling from proapoptotic stress toward induction of apoptosis.

Bcl-2 decreases the free Ca2+ concentration within the endoplasmic reticulum.

The antiapoptotic protein Bcl-2 localizes not only to mitochondria but also to the endoplasmic reticulum (ER). However, the function of Bcl-2 at the level of the ER is poorly understood. In this study, we have investigated the effects of Bcl-2 expression on Ca(2+) storage and release by the ER. The expression of Bcl-2 decreased the amount of Ca(2+) that could be released from intracellular stores, regardless of the mode of store depletion, the cell type, or the species from which Bcl-2 was derived. Bcl-2 also decreased cellular Ca(2+) store content in the presence of mitochondrial inhibitors, suggesting that its effects were not mediated through mitochondrial Ca(2+) uptake. Direct measurements with ER-targeted Ca(2+)-sensitive fluorescent "cameleon" proteins revealed that Bcl-2 decreased the free Ca(2+) concentration within the lumen of the ER, [Ca(2+)](ER). Analysis of the kinetics of Ca(2+) store depletion in response to the Ca(2+)-ATPase inhibitor thapsigargin revealed that Bcl-2 increased the permeability of the ER membrane. These results suggest that Bcl-2 decreases the free Ca(2+) concentration within the ER lumen by increasing the Ca(2+) permeability of the ER membrane. The increased ER Ca(2+) permeability conferred by Bcl-2 would be compatible with an ion channel function of Bcl-2 at the level of the ER membrane.

A mammalian H+ channel generated through alternative splicing of the NADPH oxidase homolog NOH-1.

Voltage-gated proton (H+) channels are found in many human and animal tissues and play an important role in cellular defense against acidic stress. However, a molecular identification of these unique ion conductances has so far not been achieved. A 191-amino acid protein is described that, upon heterologous expression, has properties indistinguishable from those of native H+ channels. This protein is generated through alternative splicing of messenger RNA derived from the gene NOH-1 (NADPH oxidase homolog 1, where NADPH is the reduced form of nicotinamide adenine dinucleotide phosphate).